中国西南横断山区小型兽类嗜吞噬细胞无形体基因的检测及序列测定

Identification of Anaplasma phagocytophilum in small mammals from Hengduan Mountains of Southwest China

目的了解中国西南横断山区小型兽类自然感染嗜吞噬细胞无形体的情况。方法采集位于滇西北横断山区的高黎贡山山脉、香格里拉雪山等山地（海拔1000～4500m）林区的小型兽类脏器标本以低温保存和运输，所获标本在实验室应用聚合酶链反应（PCR）方法对嗜吞噬细胞无形体16SrRNA基因和Msp4基因片段进行扩增测序，并将所测序列与GenBank中注册的基因序列进行相似性比较。结果共检测小型兽类5目18属35种共436只，从6属11种小型兽类中发现阳性标本32份，总阳性率为7．34％。其中，高黎贡山林区检测小型兽类标本25种301只，阳性26份，阳性率为8．64％（26／301）；香格里拉雪山等林区检测小型兽类标本19种135只，阳性6份，阳性率为4．44％（6／135）；阳性标本绝大部分发现于小型兽类中的啮齿类动物。序列比较分析表明：不同小型兽类间的16SrRNA基因序列相似性为99％-100％，且与吉林野鼠中检测的无形体相对应片段（GenBank：DQ449948）最相近，相似性达99％-100％。对其Msp4基因核苷酸序列进一步分析发现与GenBank中相应片段相似性为95％-97％，提示中国西南横断山区小型兽类所感染无形体株变异较大。结论首次证实和发现中国西南横断山区6属11种小型兽类自然感染嗜吞噬细胞无形体，其中啮齿类动物可能是这一地区嗜吞噬细胞无形体的主要宿主。

Objective To investigate the prevalence ofAnaplasma phagocytophilum in small mammals from the forest area of Hengduan Mountains in southwestern China. Methods Small mammals captured from Gaoligong and Xianggelila mountainous area of Yunnan province were detected by PCR amplification. The sequences of 16S rRNA and Msp4 gene fragments from positive samples were compared with corresponding sequences deposited in GenBank. Results A total number of 436 small animals, which belongs to 5 orders 18 genera 35 species were tested, 32 （7.34%） were positive in 6 genera 11 species. There were 8.64% （26/301） positive in 25 species at Goligong mountainous areas, and 4.44% （6/135） were positive in 19 species at the Xianggelila mountainous areas. Positive small mammals were most rodents. The nucleotide sequences of A.phagocytophilum 16S rRNA gene amplified from small mammals varied from 99%-100% and were 99%-100% similar with the corresponding segments of A. phagocytophilum from Jilin deposited in GeneBank. The sequences ofA. phagocytophilum Msp4 gene showed that there was 95%-97% similarity with the corresponding sequences registered in GenBank. Conclusion A. phagocytophilum was firstly identified in 6 genera 11 species small mammals from a forest area of Hengduan Mountainous areas in southwestern China. Rodents might serve as the primary hosts indicating the potential risk to the domestic animals and human beings in this area.